Expression of G protein-coupled receptor kinase 4 is associated with breast cancer tumourigenesis

Journal of PathologyJ Pathol 2008; 216: 317–327Published online 29 July 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2414

Original Paper

Expression of G protein-coupled receptor kinase 4is associated with breast cancer tumourigenesis

J Matsubayashi,1# M Takanashi,2,3# K Oikawa,2,3 K Fujita,2 M Tanaka,2 M Xu,2 A De Blasi,4 M Bouvier,5

M Kinoshita,6 M Kuroda2* and K Mukai11Department of Diagnostic Pathology, Tokyo Medical University, Tokyo, Japan2Department of Pathology, Tokyo Medical University, Tokyo, Japan3Department of Cell Therapy, Tokyo Medical University, Tokyo, Japan4Department of Experimental Medicine, University ‘La Sapienza’, Rome, Italy5Department of Biochemistry and Groupe de Recherche Universitaire sur le Medicament, Universite de Montreal, Canada6Department of Surgery, Kosei Chuo Hospital, Tokyo, Japan

*Correspondence to:M Kuroda, Department ofPathology, Tokyo MedicalUniversity, 6-1-1, Shinjuku,Shinjuku-ku, Tokyo160-8402, Japan.E-mail: [email protected]

#These authors contributedequally to this study.

No conflicts of interest weredeclared.

Received: 30 January 2008Revised: 14 June 2008Accepted: 15 July 2008

AbstractG-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian ser-ine/threonine protein kinases that phosphorylate and regulate agonist-bound, activated,G-protein-coupled receptors (GPCRs). GRKs and β-arrestins are key participants in thecanonical pathways leading to phosphorylation-dependent GPCR desensitization, endocy-tosis, intracellular trafficking and resensitization. Here we show that GRK4 isoforms areexpressed in human breast cancer but not in normal epithelia. In addition, GRK4-over-expressing cells activated the mitogen-activated protein kinase (MAPK) mediated by ERK1/2 and JNK phosphorylation in breast cancer-derived cell lines. Furthermore, suppressionof β-arrestins decreased GRK4-stimulated ERK 1/2 or JNK phosphorylations. These dataindicate that high-level expression of GRK4 may activate MAPK signalling pathways medi-ated by β-arrestins in breast cancer cells, suggesting that GRK4 may be implicated in breastcancer carcinogenesis.Copyright 2008 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: GRK4; β-arrestin; breast cancer; GPCR; MAPK

Introduction

Breast cancer is the most common malignancy amongfemales in most Western countries, where women havean overall lifetime risk of over 10% for developing it[1], and is a heterogeneous disease sustained by com-plex growth pathways. As well as hormone respon-siveness, breast tumours may show HER-2/neu and/orEGFR gene amplification and over-expression [2–4].In addition, trastuzumab is the first agent approvedfor therapy of HER-2/neu over-expressing tumours.Both targeted therapy and hormone therapy for breastcancers have significantly impacted on survival ofthe patients. However, metastatic breast cancer stillremains an incurable disease.

G protein-coupled receptor kinases (GRKs) [5] initi-ate homologous desensitization by phosphorylating thethird cytoplasmic loop or tail of activated G protein-coupled receptors (GPCRs) [5,6], thereby allowingcells to adapt to changing extracellular signals. GRKsare serine/threonine-directed protein kinases com-posed of seven isoforms divided into three fami-lies [7]. GRK1 and GRK7 compose the rhodopsin

kinase/visual family, are expressed exclusively inretina, and participate in desensitization of opsins inrods and cones [8–10]. GRK2 and GRK3 were orig-inally identified as a regulator of the β-adrenergicreceptor and compose the ARK family. On the otherhand, the GRK4 family consists of GRK4, GRK5and GRK6. Although GRKs 2, 3, 5 and 6 are verywidely distributed in mammalian tissues, GRK4 is pre-dominantly found in testis [11] and to lesser extentin Purkinje cells and kidney [12,13]. GRK-mediatedphosphorylation has been shown to decrease the recep-tor/G protein interactions and to initiate β-arrestinsbinding to the phosphorylated GPCR [14]. Arrestinsare a family of scaffolding proteins associated withthe function of GPCRs and consist of four mem-bers, arrestin 1 and arrestin 4, visual-arrestins; andarrestin 2 (β-arrestin 1) and 3 (β-arrestin 2), non-visual arrestins. Visual arrestins are expressed mainlyin the retina [15], but β-arrestins 1 and 2 are ubiqui-tously expressed in most tissues and play an impor-tant role in desensitization and internalization foractivated GPCRs [16]. In addition, β-arrestins areassociated with various signalling molecules, such

Copyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.www.pathsoc.org.uk

318 M Kuroda et al

as ERK 1/2, JNK and Src [17]. In this report wedescribe studies of GRK4 and our results suggestnovel GRK4’s cellular roles and a novel mechanismwhereby GRK4 may be implicated in human breastcancer development.

Materials and methods

Tissues

RNA samples from human breast tissues were obtainedfrom Genomics Collaborative, Inc. (Boston, MA,USA) and Kosei Chuo Hospital, and human breasttissue samples for protein analysis were obtainedfrom Kosei Chuo Hospital. This study was institu-tional review board-approved and all patients pro-vided written informed consent. The tumour samples(summarized in Table 1) were fixed in 10% neutralbuffered formalin, processed routinely and embeddedin paraffin. The histological diagnosis was confirmedby review and all tumours were re-evaluated for his-tological type and graded by two senior pathologistswho were unaware of the clinical data.

Quantitative real-time PCR analysis

First strand cDNA was synthesized from 1 µg totalRNA with Oligo (dT)17 primer and M-MLV reversetranscriptase (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was performed using the Mx3005PQPCR system (Stratagene, La Jolla, CA, USA)

with TaKaRa Ex Taq R-PCR ver. 2.1 (Takara BioInc., Shiga, Japan), ROX Reference Dye (Strata-gene) and SYBR Green I (Cambrex, Washington,DC, USA). We used GRK4-specific primers 5′-GGGACTGAAGGAGGAGAACC-3′ and 5′-TTTG-TTACGGGTTGCCTTTC-3′; HER2/neu-specificprimers 5′-TGGAGAACCCCGAGTACTTG-3′ and 5′-TAGGTGTCCCTTTGAAGGTG-3′; and EGFR-specific primers 5′-CCAAGCCATATGACGGAATC-3′ and 5′-ACTATCTGCGTCTATCATCC-3′. β-actin-specific primers were previously described [18]. Reac-tion mixtures were denatured at 95 ◦C for 30 s and thenwere subjected to 50 PCR cycles at 95 ◦C for 10 s and60 ◦C for 30 s. GRK4, HER2/neu and EGFR mRNAlevels were normalized to β-actin signals. We per-formed these experiments to determine mRNA levelsin duplicate.

Cell culture

NIH 3T3 cells were cultured in DMEM (Sigma)supplemented with 10% bovine serum (BS), andMCF-7, HMC1-8 and MRKnu-1 cells were culturedin RPMI1640 (Sigma) supplemented with 10% fetalbovine serum (FBS). For transfection of the GRK4expression vectors, MCF-7 cells were maintained inRPMI1640 medium containing 5% charcoal/dextran-treated fetal bovine serum (Hyclone, Logan, UT,USA). All the cells were maintained at 37 ◦C in a 5%CO2 environment.

Table 1. Association between GRK4 expression and patients’ characteristic

Total(n = 78)

GRK4 strongpositive (n = 30)

GRK4 weakpositive (n = 26)

GRK4absent (n = 22)

p Value strong/weakversus absent

Age≤40 6 2 1 3>40 72 28 25 19

Histological typeDuctal 68 24 25 19 NSLobular 7 5 1 1Mucinous 2 1 0 1Medullary 1 0 0 1

Histological gradeI + II 42 11 16 15 NSIII 26 13 9 4Non-invasive 10 6 1 3

Tumour size≤2 cm 36 11 14 11 NS>2 cm 42 19 12 11

Lymph node metastasisPositive 33 18 7 8 p < 0.05∗Negative 43 12 18 13Unknown 2 0 1 1

ER statusPositive (3+, 2+) 43 16 18 9 NSNegative (1+, 0) 34 14 7 13Unknown 1 0 1 0

Her2/neu statusPositive (3+, 2+) 20 10 7 3 NSNegative (1+, 0) 51 18 18 15Unknown 7 2 1 4

J Pathol 2008; 216: 317–327 DOI: 10.1002/pathCopyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

GRK4 expression in breast cancer 319

Transient expression of GRK4s

The expression vectors of the full-length GRK4α,β, γ , δ and GRK4-delKD were previously described[11,19]. For transient transfection experiments, 2 µgplasmid was transfected into MCF-7 cells usingDoFect GT1 transfection reagent (Dojindo Laborato-ries, Kumamoto, Japan) according to the manufac-turer’s recommendations.

Immunoblotting and immunoprecipitation

Cells were lysed in IP buffer (20 mM Tris–HCl, pH7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10%glycerol, with a cocktail of protease inhibitors), andthen the cell lysates were incubated with a specificanti-β-arrestin antibody (Sigma) or rabbit IgG (WakoPure Chemical, Osaka, Japan). The immune com-plexes were reacted with 50% protein G sepharose(Amersham). The protein extracts and immunoprecip-itates were suspended in SDS loading buffer. Afterboiling, equal amounts (10 µg) of proteins or immuno-precipitates were run on 15% SDS–PAGE gels andthen transferred to Immobilon membranes (Millipore)by a semi-dry blotting method. The membranes wereprobed with antibodies using standard techniques. Thesignals were visualized by ECL plus (GE HealthcareBio-Science) and detected with LAS-3000 mini (Fuji-film, Tokyo, Japan). The anti-human GRK4s (H-70;Santa Cruz Biotechnology, Santa Cruz, CA, USA) andthe anti-β-arrestin (A7977; Sigma) monoclonal anti-bodies were applied at a dilution of 1 : 500. Anti-ERK(610030; Cell Signaling), anti-phosphoERK (612358;Cell Signaling), anti-JNK (9258; Cell Signaling) andanti-phospho JNK (9258; Cell Signaling) were alsoused according to the manufacturer’s instructions.

Immunohistochemistry

Immunohistochemical assays were performed onformalin-fixed paraffin-embedded sections with Ven-tana HX System Benchmark (Ventana Medical Sys-tems). An anti- GRK4α- and β-specific antibody (K-20; Santa Cruz Biotechnology) and anti-ER antibody(Ventana, Tuscon, AZ, USA) were used. Her-2/neustatus was determined by means of immunohistochem-istry, using the Dako HercepTest (Dako Japan, Kyoto,Japan) and scored with the Dako scoring system. Sam-ples with a score of 2+ and 3+ were consideredHER2-positive; on the other hand, they were con-sidered ER-positive if 1% of cells showed nuclearstaining. The intensity of GRK4 staining was evalu-ated using the following criteria: strong positive (2+),dark brown staining in more than 50% of tumour cellscompletely obscuring the cell membrane and cyto-plasm; weak positive (1+), any lesser degree of brownstaining appreciable in the tumour cell membrane andcytoplasm; absent (scored as 0), no appreciable stain-ing in tumour cells.

In vitro proliferation assays

The effects of GRK4s and β-arrestin on the growthof MCF-7 were tested using the MTT cell growthassay kit (Cell Count Reagent SF, Nacarai Tesque,Kyoto, Japan). MTT assay was performed accordingto the manufacturer’s recommendations. The reagentswere added to each well and incubated at 37 ◦Cfor 4 h. The MTT reduced by living cells into aformazan product was assayed with a multi-wellscanning spectrophotometer at 450 nm.

In Figure 4D, MCF-7 cells were transfected withsiRNAs and then the cells were cultured and treatedwith the MTT cell growth assay reagent to testcell proliferation. Total RNAs from the cells weresubjected to quantitative real-time PCR analysis. InFigure 6, MCF-7 cells were transfected with β-arrestinsiRNAs; 2 days after the siRNAs transfection, the cellswere further transfected with GRK4 expression vectorsand plated in a 96-multiwell plate at 800 cells/well.The cell numbers were then assessed with MTT assayat 24, 48 and 72 h after vector transfection.

RNA interference

MCF-7 was transfected with 50 nM siTrio Full Setspecific for human GRK4 or β-arrestins 1, or 2(B-Bridge International Inc; Mountain View, CA,USA) with 12 µl of HiPerFect (Qiagen) reagent ineach 60 mm culture dish, according to the manufac-turer’s recommendations. The target sequences of thesynthetic oligonucleotides for siRNA against GRK4and β-arrestins 1 and 2 were as follows: GRK4#1,5′-CAAAAAAGCCUUUGAGGAA-3′; GRK4#2, 5′-GGACAGAGGGUUCGAGGAA-3′; GRK4#3, 5′-GGACAUUCUCCAUUCAAAA-3′; β-arrestin 1, 5′-AGCCUUCUGCGCGGAGAAU-3′; β-arrestin 2, 5′-GGACCGCAAAGUGUUUGUG-3′. Negative controlsiRNA (AUCCGCGCGAUAGUACGUAdTdT; syn-thesized by B-Bridge International Inc.) was also trans-fected into cells as a negative control.

Statistical analysis

Data are presented as mean ± SD. Students’ t-test wasused for comparing clinical factors between GRK4-positive and -negative tumours and significance wasset at p < 0.05.

Results

GRK4 expression in breast cancer

Using quantitative real-time PCR analysis, we foundhigh-level expression of the GRK4 mRNA in invasivelobular and ductal carcinomas compared with normalbreast tissues (Figure 1). HER2/neu and epidermalgrowth factor receptor (EGFR) is genetically amplifiedand over-expressed in aggressive breast cancers. Theexpression pattern of the GRK4 mRNA, however,


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Figure 1. Quantitative real-time PCR analysis demonstratingGRK4, HER2/neu and EGFR mRNA levels in breast cancer andnormal breast tissues. β-Actin was used as an internal controlfor mRNA quantity. The minimum mRNA expression level wasarbitrarily set to 0.1 in the graphical presentation; all othermRNA signals were normalized to this value. Bars, SD

was different from that of the HER2/neu and EGFRmRNAs, as shown in Figure 1.

Expression of GRK4 isoforms

The GRK4 subfamily has four alternative splicevariants, named GRK4α, β, γ and δ [11,20,21](Figure 2A). To investigate the expression pattern ofthe GRK4 subfamily, we performed western blot anal-ysis of representative breast cancer, fibroadenoma andnormal tissue samples, using an anti GRK4 anti-body that reacts with all GRK4 isoforms. We foundthat the GRK4 subfamily expression in five cases ofinvasive ductal carcinoma (IDC), while GRK4s werehardly detectable in normal breast tissues (Figure 2B,C). Interestingly, Cases 35 and 42 (IDC) mainlyexpressed GRK4β and GRK4δ, but Cases 3 and25 (IDC) expressed GRK4β, GRK4γ and GRK4δ

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Figure 2. Structures of GRK4 isoforms and their expressionsin breast cancer tissues. (A) Diagram of the gene products ofthe human GRK4 splicing variants. The diagram represents theamino acid sequence of the four GRK4 isoforms that resultfrom alternative splicing of the GRK4 mRNA. GRK4 delKD isa deletion mutant form of GRK4. (B) Western blot analysis ofGRK4 protein from breast cancer, fibroadenoma, and normalbreast tissues. GRK4α (lane 1), β (lane 2), γ (lane 3) or δ (lane4)-over-expressing NIH3T3 cells are also shown as positivecontrols. (C) Expression of GRK4-isoforms in breast cancertissues; 15 samples of breast cancer and three samples ofnormal tissues were analysed by western blotting

(Figure 2B), suggesting that breast cancer may showvarious expression patterns of the GRK4 subfamily.

Unfortunately, the antibody used in Western blot-ting was not applicable to immunohistochemistry.Therefore we used another antibody specific only forGRK4α and β. It should be noted that GRK4α isthe full-length version and the most homologous withother GRKs among four isoforms of GRK4. GRK4α,but not the other GRK4 isoforms, phosphorylatesdopamine receptor [22] and rhodopsin [11]. We classi-fied the expression patterns of GRK4α and/or β in thebreast cancer tissue samples in the range of absent tostrong positive (Figure 3). Of the 78 cases examined,GRK4α and/or β were strongly stained in 30 cases(38.5%; score 2+), weakly stained in 26 cases (33.3%;score 1+) and not stained in 22 cases (28.2%; score0) (details are shown in Table 1). Immunohistochemi-cal analysis of breast tumour tissues demonstrated that



H&E H&ECase 55

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Figure 3. Immunohistochemical analysis of GRK4 expression in breast cancer tissues. Left panel is haematoxylin and eosin-stainedand right panel is GRK4α and/or β in each case. We evaluated the intensity of GRK4 staining as absent (Case 55), weak positive(Case 70) and strong positive (Cases 1, 3, 25 and 38). ∗, normal myoepithelium of mammary gland; arrow head, normal epitheliumof mammary gland

positive staining for GRK4α and/or β seemed to bespecific for cancer cells and was hardly detectablein normal myoepithelium (Figure 3, Case 38) andepithelial cells (Figure 3, Case 1) of the mammarygland. Both invasive ductal carcinoma and lobular car-cinoma specimens showed cytoplasmic-localized ofGRK4α and/or β (Figure 3). Furthermore, we eval-uated the biological and clinicopathological signifi-cance of GRK4 expression. As shown in Table 1, wefound that lymph node metastasis (higher in positive;p < 0.05 by Students’ t-test) is significantly associ-ated with GRK4 expression (score 1 + –2+). We alsoexamined the expression of GRK4α and/or β proteinsin normal human tissues by means of tissue microar-rays containing all normal human tissues, and foundthat GRK4α and/or β proteins were expressed in Purk-inje cells of cerebellum, seminiferous epithelium oftestis and renal tubule of kidney (data not shown).These results were consistent with previous reports[11–13].

Effects of GRK4s on cell proliferation activity

Because we observed high-level expression of GRK4in breast cancers, we sought to determine whetheror not GRK4 over-expression promotes tumour cellproliferation. We transiently transfected MCF-7 cellswith a GRK4α, δ or GRK4 construct missing its cen-tral catalytic domain (�173–445; GRK4-delKD) [19]expression vector, respectively (Figure 4A). Usingthe MTT assay, we found that the cells over-expressing GRK4α and δ showed increased growth

rates compared with the cells transfected with a con-trol vector (Figure 4B). In contrast, no significantchange was observed in the proliferation of GRK4-delKD over-expressing cells. These results suggestthat over-expression of GRK4 may play an importantrole in breast carcinogenesis and affect tumour cellproliferation.

GRK4 siRNA suppresses the growth of breastcancer cells

To examine the effects of GRK4 repression in breastcancer cells, we treated a breast cancer-derived cellline, MCF-7, with the mixture of three 21-nucleotidedouble-stranded small interfering RNAs (siRNAs) spe-cific for the GRK4 variants. The siRNAs reducedGRK4 expression in the cells, and suppressed thegrowth of the cells significantly (Figure 4C, D). Thesedata suggested that up-regulation of GRK4 expressionis concerned with growth or survival of breast can-cer cells, and GRK4 may become a novel therapeutictarget for breast cancer treatment.

GRK4 regulates MAP kinase cascade

Previous studies revealed that MAPK signalling path-ways with the EGF family, including EGFR, ErbB2and ErbB3 or ER, are important for cancer devel-opment and progression of breast cancers [23–26].Thus, we examined whether GRK4 expression acti-vates MAPK signal transduction in breast cancer cells.Because MAPK in breast cancer-derived cell lines


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Figure 4. Effects of GRK4s on cell proliferation activity. (A) Western blotting analysis of MCF-7 cells that were transfected withGRK4α, δ and delKD expression vectors and a control vector (pcDNA). (B) MCF-7 cells were transfected with GRK4α, δ anddelKD expression vectors and a control vector (pcDNA), and then cell numbers were analysed with MTT assays at several timepoints, as described in Materials and methods. Bars, SD. (C) Reduction of the GRK4 transcript level by the active siRNA in MCF-7cells. 48 h after siRNA transfection, the MCF-7 cells were harvested. Total RNA was extracted from the cells and subjected toreal time PCR analysis. GRK4, active siRNA specific for GRK4; Cont, non-silencing negative control siRNA. Data were normalizedto the mRNA level of control in MCF-7 that was arbitrarily set to 1 in the graphical presentation. Bars, SD. (D) Active siRNAspecific for GRK4 inhibits cell growth. MCF-7 cells transfected with the active siRNA or negative control siRNA were harvestedat 0, 24, 48 and 72 h after transfection. The cell numbers were analysed by MTT assays. Bars, SD

are usually activated by factors such as EGF or ERin FCS in a culture medium, we established MCF-7 cells possessing inactivated MAPK by growingin a medium with charcoal/dextran-treated FCS for6 months, according to Oesterreich et al [27]. Then weintroduced expression vectors carrying GRK4s or thedeletion mutant form GRK4-delKD into the MCF-7cells by transfection. 24 h after transfection, we exam-ined the expression and phosphorylation of ERK1/2and c-Jun amino-terminal kinase (JNK), and found thatERK1/2 were phosphorylated in cells over-expressingGRK4α, β or γ compared with the cells transfectedwith the control vector, a GRK4–delKD expressionvector or a GRK4δ expression vector. Phosphorylationof JNK was detected in all cells expressing one of theGRK4 isoforms or GRK4–delKD (Figure 5A). Theseresults indicate that GRK4 induces MAPK signalling

activation through ERK1/2 and JNK signalling path-ways in breast cancer cells.

The phosphorylation of G protein-coupled recep-tors (GPCRs) by GRK4 allows the recruitment ofβ-arrestin that binds to the GPCR and initiates its inter-nalization. β-Arrestin is a cytosolic protein that medi-ates activated-GPCR desensitization and contributesto GPCR signalling as an adaptor or a scaffold forthe recruitment of signalling molecules into complexwith agonist-occupied receptors [17,28]. The mitogen-activated protein and extracellular signal-regulatedkinases (MEK)-ERK1/2 cascade is a commonly acti-vated in breast cancer and important component ofEGF–EGFR signalling pathways. β-Arrestin inter-acts with molecules such as Src, Raf, Erk, ASK1and JNK3 and regulates several pathways resultingin the activation of MAPK [29–32]. Therefore, toexamine whether GRK4 involved β-arrestin signalling



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Figure 5. GRK4 interacted with β-arrestin and activated ERK 1/2 signalling pathways. (A) Effects of GRK4 over-expression onthe ERK 1/2 signalling pathway. MCF-7 cells were transfected with GRK4α, β , γ , δ, delKD expression vectors or a controlvector (pcDNA), as indicated, and then the cells were analysed by western blotting, using phospho-ERK 1/2, total ERK 1/2,phospho-JNK and total JNK antibodies. Columns represent the ratios of phospho-ERK1/2 per total ERK1/2, and phospho-JNKper total JNK, respectively. Data were normalized to the ratio of the vector control, which was arbitrarily set to 1 in the graphicalpresentation. (B) HMC1-8 and MRK nu-1 cells were subjected to immunoprecipitation with a β-arrestin antibody, and thenthe immunoprecipitates were analysed by western blotting with an anti- GRK4 antibody. IP, immunoprecipitation; WB, westernblotting

pathways, we performed immunoprecipitation analysisusing human breast cancer-derived cell lines HMC1-8 and MRKnu-1 in which GRK4 is expressed at ahigh level. Unfortunately, anti-GRK4 specific antibod-ies examined in this study were not applicable forimmunoprecipitation. However, immunoprecipitationusing an antibody specific for both β-arrestin 1 and2 demonstrated that β-arrestins form complexes withGRK4α and β (Figure 5B). Thus, GRK4s can existin a complex with β-arrestins in human breast cancercell lines.

GRK4 regulates MAP kinase cascade via β-arrestinin breast cancerThese data suggest a possible role of β-arrestins inthe GRK4-mediated activation of MAPK signalling.Therefore, we examined whether or not the knock-down of β-arrestin 1 or 2 prevents MAPK signalling.

We introduced siRNAs specific for β-arrestin 1 and 2,respectively, into the MAPK-inactivated MCF-7 cellsby transfection; 2 days after the siRNA transfection,we further introduced the expression vectors carry-ing GRK4α, δ and GRK4-delKD, respectively. Thenwe examined the expression and phosphorylation ofERK1/2 and JNK at 24 h after transfection. We founddecreased phosphorylation of ERK1/2 and JNK in bothGRK4α and δ over-expressing cells with β-arrestin 1siRNA (Figure 6A). On the other hand, no significantchange of phosphorylation state was detected in cellswith β-arrestin 2 siRNAs (Figure 6A).

Furthermore, we examined whether or not β-arrestin1 and 2 siRNAs prevents tumour cell growth in cellsover-expressing GRK4 isoforms. We found that bothβ-arrestin 1 and 2 suppressed the growth of cellsover-expressing GRK4α or δ (Figure 6B). These datasuggest that β-arrestin may play important roles in the


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Figure 6. Knockdown of β-arrestin prevents MAPK signalling activation and tumour cell growth. (A) MCF-7 cells were transfectedwith β-arrestin 1 or 2 siRNA, or control siRNA, as indicated in Materials and methods, and then the cells were transfected withGRK4α, δ or delKD expression vectors or a control vector (pcDNA). After transfections, cell were analysed by western blottingwith phospho- or total ERK 1/2 and phospho- or total JNK antibodies. Columns represent the ratios of phospho-ERK1/2 per totalERK1/2, and phospho-JNK per total JNK, respectively. Data were normalized to the ratio of vector control that was arbitrarily setto 1 in the graphical presentation. (B) MCF-7 cells were transfected with β-arrestin 1 or 2 siRNA or control siRNA as indicated,and then the cells were transfected with GRK4α, δ or delKD expression vectors or a control vector (pcDNA). Cell numberswere analysed by MTT assays. Bars, SD

GRK4-mediated activation of MAPK module and cellgrowth.

Discussion

GRK4 expression has previously been reported tobe restricted to lung, kidney, brain and testis among

normal tissues [33]. GRKs have been thought to playin the transmission of light and odorant signals, themediation of hormone action and high-level expressionor polymorphisms of GRK4 associated with hyper-tension [34]. In this study, we have first revealedthat GRK4 isoforms are expressed in breast cancertissues with various expression patterns, but not in



normal mammary glands (Figure 3). GRK4 plays animportant role in agonist-activated G protein-coupledreceptors (GPCRs) signalling by phosphorylation atthe C-terminus of GPCRs to desensitization and inter-nalization. Therefore, because of high-level expres-sion of GRK4 in breast cancer, some GPCRs maybe involved in breast cancer development and/or pro-gression. In addition, cell proliferation assay withGRK4 knockdown (Figure 4) in this study proposesthe notion that GRK4 may be relevant to carcinogen-esis in breast cancer. Thus, these results may shedlight on novel functional features of breast cancercarcinogenesis.

The GRK4 gene undergoes alternative RNA splic-ing. The resultant GRK4 isoforms have a ser-ine/threonine kinase catalytic domain in the centre anda regulator of G-protein signalling (RGS)-like domainwithin the N-terminus region (Figure 2A). Thesedomains regulate GPCR signalling via phosphory-lation-independent mechanisms. Thus, GRK4s inbreast cancer may regulate phosphorylation and/or aserine/threonine kinase in MAPK signalling, becauseMAPK is activated by ER, EGFR or ErbB2 and reg-ulates tumour growth of breast cancer. We detectedphosphorylation of ERK and JNK in GRK4-over-expressing cells but not in cells transfected with a vec-tor control or a GRK4–delKD deletion mutant expres-sion vector (Figure 5A), suggesting that the catalyticdomain, but not RGS in GRK4s, activates MAPK sig-nalling via ERK and JNK. In addition, transfection ofa GRK4 expression vector into MCF-7 cells inducedcell proliferation compared with the cells transfectedwith a vector control or a GRK4–delKD expressionvector (Figure 4A). Thus, the kinase catalytic domainof GRK4 may be important for tumour growth.

Next, we considered how GRK4 activates theMAPK signal transduction, and hypothesized thatGRK4 indirectly activates MAPK signalling mediatedby some molecules in breast cancer cells. Previousstudies have revealed that co-expression of GRK4and the D1 dopamine receptor in a heterologous sys-tem induces phosphorylation of the receptor in theabsence of agonist, causing constitutive desensitizationand internalization of the receptor [22]. For a num-ber of GPCRs, a rapid, drastic and reversible loss ofresponsiveness has been shown to occur upon expo-sure to agonists (homologous desensitization). Fromthe point of a physical function, β-arrestin is a can-didate for the molecule mediating MAPK signalling.This molecule is deeply concerned with GRK4 in theGPCRs signalling pathway. Typically, GRK4 phos-phorylates agonist-activated GPCRs, initiating theirhomologous desensitization and internalization withits functional co-factor β-arrestin [19,35–37]. Uponactivation of angiotensin II type 1A [30], neurokinin1 [38] and the protease-activated [39] receptors, β-arrestin scaffolds the components of the extracel-lular signal-regulated kinase (ERK) cascade, Raf-1,MEK1 and ERK1/2 into a complex with the recep-tors, leading to the activation of ERK1/2 [32]. In

addition, Kim et al reported that over-expression ofGRK 5 or 6 enhances β-arrestin-mediated ERK acti-vation [32]. In this study, we confirmed that β-arrestinplays an important role in GRK4-mediated activationand induces the MAPK signalling pathway throughERK1/2 and JNK (Figure 6). Interestingly, both β-arrestin 1 and 2 siRNAs suppressed cell proliferationof the GRK4s-over-expressing cells (Figure 6B). Onthe other hand, only the β-arrestin 1 siRNA inhibitedphosphorylation of ERK and JNK in the GRK4-over-expressing cells (Figure 6A). The difference betweenβ-arrestin 1 and 2 functions in breast carcinogenesis isstill elusive. Noble et al reported that the N-terminaldomain and the conformation of β-arrestin 1 in theactivated state was different from that of β-arrestin 2[40]. It might be possible that β-arrestin 1 and 2 aredifferent in the functions of signal transduction foragonist-occupied receptor sensitization and internal-ization. The co-immunoprecipitation assay suggestedthat GRK4 is associated with β-arrestin (Figure 5B).Therefore, ectopic expression of GRK4 may directlylead to an increasing β-arrestin-mediated MAPK sig-nalling activation that promotes cell growth.

GRK4 expression level was likely to be associatedwith lymph nodes metastasis (Table 1). This is, to ourknowledge, the first study to show the prognostic valueof GRK4 expression in human cancers. The detailedmolecular mechanism of GRK4 with cancer metas-tasis is still unknown. A recent study has revealedthat β-arrestin regulates oncoprotein Mdm2 [41]. Fur-thermore, Mdm2 regulates E-cadherin degradation bybinding to β-arrestin [42]. Thus, we considered thatectopic expression of GRK4 may regulate E-cadherindegradation through the β-arrestin–Mdm2 complex bya ubiquitin proteasome lysis system, although furtherstudies are required to confirm this speculation.

GPCRs represent a major target for therapeuticagents, and the continuing identification of orphanGPCRs offers opportunity for future pharmacolog-ical and therapeutic development [43]. The recentadvancement of molecular technologies has allowedfor a better understanding of the mechanism sustainingbreast cancer therapy. Molecular targeting compoundsand hormone therapy have significantly impacted onthe survival of these patients. However, therapy ofmetastatic disease still remains palliative and alsoadjuvant treatments do not guarantee optimal results.GRK4 siRNA suppressed the growth of breast cancercells (Figure 4B). GRK4 is considered to participate inGPCRs signalling. Therefore, the orphan GPCR regu-lated by GRK4 may become an excellent therapeutictarget of breast cancer. In fact, GRK4 expresses onlyin testis, Purkinje cells and kidney, whereas GRK2,3, 5 and 6 are widely expressed in human normaltissues [12,13]. The aim of molecular-targeted ther-apy is suppression of the transformed phenotype withminimal effects on normal cells. In addition, upstreamof GRK4 signalling might be different from knownHER2/neu and EGFR signalling. Therefore, we expect


326 M Kuroda et al

that GRK4 is one of candidates as a target for breastcancer therapy.

In summary, GRK4 may be involved in breastcancer carcinogenesis. GPCR targeting for GRK4may also be concerned with breast cancer. Theseresults might offer a novel target for pharmacologicalintervention.

Acknowledgements

We thank George Horio, Shigeki Hamano, Tomotoshi Nagama,Misayo Kimura, Ayumi Akiyoshi, Hiroake Iobe, Shunji Hasebeand Kazuhiro Mori for technical assistance and Yuki Hasegawafor outstanding editorial assistance. This study was supportedby Grants-in-Aid from the Ministry of Education, Culture,Sports, Science and Technology (MEXT) of Japan, the Min-istry of Health, Labour and Welfare of Japan, the Japan HealthSciences Foundation, a grant from Yamaguchi EndocrineResearch Association, a grant from the ‘University–IndustryJoint Research Project’ for private universities: matching fundsubsidy from MEXT (2007–2009) and Tokyo Medical Univer-sity Cancer Research Foundation.

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Expression of G protein-coupled receptor kinase 4 is associated with breast cancer tumourigenesis